CN115322965B - Method for obtaining hindbrain floor cells in vitro, complete set of culture medium and application - Google Patents
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Abstract
The invention discloses a method for obtaining hindbrain floor cells in vitro, a complete set of culture medium and application, wherein the method comprises the following steps: s1, single-layer adherence culture of undifferentiated embryonic stem cells; s2, adding a TGFbeta/Wnt/node signal channel inhibitor and a BMP signal channel antagonist into an NIM culture medium on day 0; s3, on day 1, adding a TGFbeta/Wnt/node signal path inhibitor, a BMP signal path antagonist, a Hedgehog signal path Smoothened protein agonist and a homeobox gene agonist into an NIM culture medium; s4, on the 5 th day, adding a BMP signal channel antagonist, a Hedgehog signal channel Smoothened protein agonist and a homeobox gene agonist into a NIM culture medium; s5, adding BMP signal channel antagonists and homeobox gene agonists into the NIM culture medium on the 7 th day of differentiation; s6, continuing to culture until the cells are differentiated into postbrain floor cells; the invention provides a method for obtaining hindbrain floor cells in vitro, a complete set of culture medium and application, wherein the method has high differentiation efficiency and quick differentiation time, and the differentiated hindbrain floor cells can functionally secrete SHH protein.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a method for obtaining hindbrain floor cells in vitro, a complete set of culture medium and application.
Background
The human floor FP cells are generated from the dorsal ectoderm, near the spinal chord (notochord), in the dorsal ventral region of the human brain after maturation, in the most ventral region of the human brain after maturation, with the antero-posterior axis distributed throughout the whole brain, extending from the spinal cord through the midbrain to the end brain just opposite the anterior nerve cortex, as described in the literature (Katoh, katoh M.WNT signaling pathway and stem cell signal ing network.Clin cancer Res.2007Jul 15;13 (14): 4042-5.doi:10.1158/1078-0432.CCR-06-2316.PMID: 176134527.) and (Hatta K.K.K.K.K., hork, walker C. The cyclops mutation blocks specificationof the floor plate of the zebrafish central nervous system.Nature.1991Mar28;350 (6316): 339-41.doi:10.1038/350339a0.PMID: 2008111). FP as a precursor cell for the differentiation of midbrain dopamine neurons, in vitro differentiated FP cells are also used as further differentiation of dopaminergic neurons, thereby functioning in forming the correct neural loop. Literature (Farano C A, chambers SM, lee G, tomishima MJ, studio L.effect derivation offunctional floor plate tissue from human embryonic Stem cells cell Stem cell 2010Apr2;6 (4): 336-47.doi:10.1016/j.stem.2010.03.001.PMID:20362538; PMCID: PMC 4336800.) states that abnormalities in FP cells can cause a variety of brain dysplastic diseases including forebrain uncracked deformity, microeye deformity, split plate syndrome, golin syndrome, etc., thus establishing a technical means for efficiently obtaining FP cells in vitro and providing a therapeutic means for related diseases.
The literature (Reubinoff BE, pera MF, fong CY, troenson A, bongso A. Embryonic stem cell linesfrom human blastocysts: somatic differentiation in vitro. Nat Biotechnol.2000Apr;18 (4): 399-404.Doi:10.1038/74447.Erratum in:Nat Biotechnol 2000May;18 (5): 559.PMID: 10748519.) states that human embryonic stem cell lines were isolated from human blastocysts early in 1998, and their self-renewal capacity and multipotent differentiation made them the basis for in vitro studies of human diseases, development, etc., were edited at the human embryonic stem cell level by gene editing means, and also provided ideas for the regulation of gene expression to optimize the in vitro differentiation strategy of functional cells, further disease treatment, etc.
Literature (Fananoc A, chambers SM, lee G, tomishima MJ, student L.effector derivation offunctional floor plate tissue from human embryonic Stem cells cell Stem cell 2010Apr2;6 (4): 336-47.doi:10.1016/j.stem.2010.03.001.PMID:20362538; PMCID: PMC 4336800.) provides a classical method of bi-directional inhibition SMAD (dual SMAD) of the signal pathway to establish differentiation from human embryonic Stem cell lines to neural precursor cells, by adding Noggin and SB431542, inhibiting the SMAD signal pathway to induce human embryonic Stem cells to nerves, and adding SHH on the first day of differentiation, 65% FOXA2 positive cells can be obtained on day 11. The literature (Dodd J, jessell TM, placzek M.the when and where of floor plate induction. Science.1998Nov27;282 (5394): 1654-7.doi:10.1126/science.282.5394.1654.PMID: 9867664.) describes that the FP cell-specific expression wing helix transcription factor FOXA2, the FP cell-specific molecular marker is FOXA2.
The disadvantages of the various methods reported to date for differentiating human basal cells using human embryonic stem cells are mainly: classical differentiation means requires the addition of high concentrations of protein SHH on the first day of differentiation, which has the disadvantage of being expensive and of being unstable in concentration due to production batches and preservation means, which can lead to instability in the efficiency of differentiation to FP.
Disclosure of Invention
The invention provides a method for obtaining hindbrain floor cells in vitro, a complete set of culture medium and application, wherein the method has high differentiation efficiency and quick differentiation time, and the differentiated hindbrain floor cells can functionally secrete SHH protein.
To achieve the purpose, the invention provides the following technical scheme:
in a first aspect of the invention, there is provided a method of obtaining hindbrain floor cells in vitro comprising the steps of:
s1, single-layer adherence culture of undifferentiated embryonic stem cells;
s2, adding a TGFbeta/Wnt/node signal channel inhibitor and a BMP signal channel antagonist into an NIM culture medium on the 0 th day of differentiation;
s3, changing a culture medium on the 1 st day of differentiation, and adding a TGFbeta/Wnt/node signal path inhibitor, a BMP signal path antagonist, a Hedgehog signal path Smoothened protein agonist and a homeobox gene agonist into the NIM culture medium;
s4, changing a culture medium on the 5 th day of differentiation, and adding a BMP signal channel antagonist, a Hedgehog signal channel Smoothened protein agonist and a homeobox gene agonist into the NIM culture medium;
s5, changing a culture medium on the 7 th day of differentiation, and adding a BMP signal channel antagonist and a homeobox gene agonist into the NIM culture medium;
s6, culturing is continued until the cells are differentiated into postbrain floor cells.
Preferably, the embryonic stem cells are mammalian embryonic stem cells, and more preferably, the embryonic stem cells are human embryonic stem cells.
In the present invention, the medium is changed once daily during the cell differentiation process, and the amount of the medium used is increased daily with the increase of the cell amount in the later period of differentiation. In step S1, the embryonic stem cell medium is E8 medium.
Preferably, the TGFbeta/Wnt/Nodal signaling pathway inhibitor comprises SB431542.
Preferably, the concentration of SB431542 in the medium is 8-12. Mu.m. Further preferably, the concentration of SB431542 in the medium is 10. Mu.m.
In the invention, SB431542 is a TGFbeta/Wnt/Nodal signal channel inhibitor, and is used for improving the efficiency of inducing human embryonic stem cells to differentiate into nerves in a system.
Preferably, the BMP signaling pathway antagonist comprises DMH1.
Preferably, the concentration of DMH1 in the medium is 1-2. Mu.m. Further preferred, the concentration of DMH1 in the medium is 1.5-2 μΜ; still more preferably, the concentration of DMH1 in the medium is 1.5 μΜ or 2 μΜ.
In the present invention, DMH1 is an inhibitor of selective BMP receptor and is used in the system to increase the efficiency of inducing neural differentiation of human embryonic stem cells.
Preferably, the Hedgehog signaling pathway Smoothened protein agonist comprises purporthamine.
Preferably, the concentration of Purmorphamine in the medium is 1.5-2.5. Mu.M. Further preferably, the concentration of Purmorphamine in the medium is 2. Mu.M
In the present invention, purmorphamine is a smoothened receptor agonist, activates SHH signaling pathway, and is used for improving the efficiency of human embryonic stem cell differentiation to the ventral side.
Preferably, the homeobox gene agonist comprises retinoid acid.
Preferably, the concentration of Retinoic acid in the culture medium is 1.5-2.5 μΜ. Further preferably, the concentration of Retinoic acid in the medium is 2. Mu.M
In the present invention, retinoic acid, is a ligand of Retinoic acid receptor and Retinoid X receptor for inducing differentiation of human embryonic stem cells to the posterior side.
Preferably, in step S6, the incubation time is 10-14 days. Further preferably, the cultivation time is 11 to 12 days.
Preferably, the hindbrain floor cells obtained in step S6 are capable of secreting SHH protein.
SHH protein is a secreted glycoprotein factor that can regulate proliferation, differentiation and axon formation of neural cells in embryonic and nervous system development, development and differentiation.
Preferably, after 11 days of culture, FOXA2 positive cells are 94.6±12.5%.
In a second aspect of the present invention, there is provided a kit of culture media for inducing embryonic stem cells into hindbrain floor cells in vitro, comprising a first culture medium, a second culture medium, a third culture medium, and a fourth culture medium.
The first culture medium comprises NIM culture medium, TGFbeta/Wnt/Nodal signaling pathway inhibitor and BMP signaling pathway antagonist;
the second culture medium comprises NIM culture medium, TGFbeta/Wnt/node signal channel inhibitor, BMP signal channel antagonist, hedgehog signal channel Smoothened protein agonist, and homeobox gene agonist;
the third culture medium comprises NIM culture medium, BMP signal pathway antagonist, hedgehog signal pathway Smoothened protein agonist, homeobox gene agonist;
the fourth medium comprises NIM medium, BMP signaling pathway antagonists, homeobox gene agonists.
Preferably, the TGFbeta/Wnt/node signaling pathway inhibitor comprises SB431542, and the concentration of SB431542 in the culture medium is 8-12 μΜ.
Preferably, the BMP signaling pathway antagonist comprises DMH1, the concentration of DMH1 in the culture medium is 1-2 μΜ.
Preferably, the Hedgehog (Hh) signaling pathway Smoothened protein agonist comprises purporthamine at a concentration of 1.5 to 2.5 μm in the culture medium.
Preferably, the homeobox gene agonist comprises Retinoic acid, and the concentration of the Retinoic acid in the culture medium is 1.5-2.5 mu m.
In a third aspect, the invention provides applications of the hindbrain floor cells prepared by the method in-vitro research on nerve loop constitution, treatment of various brain dysplasia diseases caused by FP cell abnormality and expansion of in-vitro differentiation brain cell methods.
Compared with the prior art, the invention has the beneficial effects and remarkable progress that: the method for obtaining the hindbrain floor cells in vitro provided by the invention is applied, and the hindbrain floor cells which functionally secrete SHH can be obtained by utilizing an embryonic stem cell system to induce differentiation for 11 days, so that the differentiation stability and the differentiation efficiency are improved. The invention can rapidly obtain the functional Floor Plate cells secreting SHH, and the differentiated Floor Plate cells are applied to the in vitro research of nerve loop constitution, the treatment of various brain dysplasia diseases caused by FP cell abnormality and the expansion of in vitro differentiation brain cell methods.
Drawings
In order to more clearly illustrate the technical solution of the present invention, a brief description will be given below of the drawings that are required to be used for the embodiments of the present invention.
It is obvious that the drawings in the following description are only drawings of some embodiments of the present invention, and that other drawings may be obtained from these drawings without inventive faculty for a person skilled in the art, but these other drawings also fall within the drawings required for the embodiments of the present invention.
FIG. 1 is a schematic diagram of the culture system during differentiation of hESC to FP in example 1 of the present invention;
FIG. 2A is an immunofluorescence of FP cells with FOXA2+ as a differentiated cell of example 3 of the present invention;
FIG. 2B is an immunofluorescence of FP cells with FOXA2+ as a differentiated cell of example 3 of the present invention;
FIG. 2C is an immunofluorescence of FP cells with FOXA2+ as a differentiated cell of example 3 of the present invention;
FIG. 2D is the result of counting FOXA2+ FP cells of example 3 of the present invention;
FIG. 3A is an immunofluorescence of the hindbrain marker HOXB1 of example 3 of the present invention;
FIG. 3B is an immunofluorescence of the hindbrain marker HOXB4 of example 3 of the present invention;
FIG. 3C is an immunofluorescence of the front midbrain marker OTX2 of example 3 of the present invention;
FIG. 4A is a schematic representation of the brain neural precursor cells of example 4 of the present invention differentiated to 11 days of cell culture supernatant culture.
FIG. 4B shows the mRNA expression level of NKX6.1 of example 4 of the present invention;
FIG. 4C shows the mRNA expression level of GLI2 of example 4 of the present invention.
Detailed Description
In order to make the purposes, technical solutions, beneficial effects and significant improvements of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.
It is apparent that all of the described embodiments are only some, but not all, embodiments of the invention; all other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
It is to be understood that:
the term "differentiation" refers to a process in which cells of the same origin gradually develop cell populations having different morphological and functional characteristics, and as a result, cells differ spatially from each other, and the same cells differ from their previous state in time. The nature of cell differentiation is the selective expression of the genome in time and space, with the turning on or off of the expression of different genes, ultimately producing a marker protein.
The term "culture medium" refers to an artificially formulated nutrient for the growth and maintenance of microorganisms, plant tissues and animal tissues, which generally contains carbohydrates, nitrogen-containing substances, inorganic salts (including trace elements), vitamins, water, and the like.
The term "signal pathway" refers to the phenomenon whereby a signal conveys a message from the outside of a cell to the inside of the cell upon which the cell is to react when a reaction is to occur in the cell. The signal pathway refers to a series of enzymatic reaction pathways that can transmit extracellular molecular signals into cells through cell membranes to exert effects. These extracellular molecular signals (called ligands) include hormones, growth factors, cytokines, neurotransmitters, and other small molecule compounds, among others.
The term "antagonist" refers to a drug that has only a strong affinity for the receptor and no intrinsic activity, and therefore does not exert an effect, but blocks the binding of an agonist to the receptor, thus antagonizing or abrogating the effect of the agonist.
The term "agonist" refers to a molecule that enhances the activity of another molecule, a drug that promotes a response, an enzyme agonist, and a hormone.
The specific meaning of the above terms in the present invention can be understood by those of ordinary skill in the art according to the specific circumstances.
It should also be noted that the following embodiments may be combined with each other, and the same or similar concepts or processes may not be described in detail in some embodiments.
The following describes the technical scheme of the present invention in detail by using specific examples.
EXAMPLE 1 culture of human embryonic Stem cell line H9
Human embryonic stem cell line H9 (H9 based on MTA number: wiCell Agreement No:21-W0349) was cultured in Matrigel coated well plates, with E8 medium, daily medium change, about 7 days to 80% confluency, either for 1:12 passage expansion or cryopreservation.
The culture medium is washed off by DPBS, then the digestion solution ReLeSR is added into the hole, the digestion solution is sucked in 1 minute, the digestion solution is digested for 7 minutes at room temperature, and an appropriate amount of E8 is added to stop the digestion and simultaneously the hole plate is beaten, so that the culture medium is preparedThe cells were beating and detached to obtain a cell diameter of about 0.1cm 2 The large and small cell clusters are passaged 1:12 into a new pore plate, or the prepared frozen stock solution (E8: FBS: DMS0=7:2:1) is added to collect cells to a frozen stock tube, the cells are transferred to a refrigerator with the temperature of-80 ℃ for pre-cooling treatment for 24 hours in the frozen stock box, and then the cells are transferred to a liquid nitrogen tank for long-term storage.
EXAMPLE 2 cultivation of H9 cell lines Using NIM Medium to give functional human basal lamina cells
2.1 coating of differentiated cell culture plates
Cell culture plates or round slides for cell differentiation were pre-coated with 0.05mg/mL Poly-L-Ornithine (ROO), i.e., polyornithine, and coated with Laminin diluted 1:50 in DMEM/F12 prior to use, and incubated at 37℃for 1 to 2 hours.
2.2 preparation of differentiated cells
H9 cells prepared in example 1 were cultured in Matrigel coated well plates using E8 until cell density reached about 80%, slesr digested, passaged 1:6 (passaging procedure as in example 1), to PO-Laminin coated well plates using NIM medium for 11 days, the NIM medium culture procedure being shown in fig. 1, small molecule compound 10 μm SB431542,2 μm DMH1 were added to NIM medium on day 0 of differentiation; 2. Mu.M Purmorphamine and 2. Mu.M Retinoic acid were added on day 1 of differentiation; SB431542 was removed on day 5 of differentiation; purmorphamine was withdrawn on day 7 of differentiation.
Example 3 immunofluorescence assay
Experimental samples:
in example 2, cells on day 11 were differentiated for detection of the expression of the FP specific marker FOXA2.
In example 2, 11 days of cells were differentiated for detection of the expression of the hindbrain markers HOXB1 and HOXB4, the foremidbrain marker OTX 2.
The experimental method comprises the following steps:
3.1 cell sample treatment
Cells to be detected were passaged onto Matrigel coated circular slides, cultured overnight to allow the cells to settle and attach to the slide surface, treated with 4% PFA fixed cells for 30 minutes, and then DPBS washed off excess PFA three times for 5 minutes each.
3.2 sealing
Blocking solution was prepared at a volume of 100 μl per sample, and serum (donkey serum or sheep serum, selected from the group consisting of serum from primary antibody sources) was 0.2% triton-dpbs=1:9, and slides were added to the blocking solution and blocked at room temperature for 30 minutes.
3.3, antibody incubation and sealing sheets
Preparing a primary antibody mixed solution and a secondary antibody mixed solution according to the volume of 100 mu L of each sample, wherein the primary antibody mixed solution is 5% serum, primary antibodies with different dilution ratios and 0.2% Triton-DPBS which is added to the required volume, and the primary antibody incubation condition is 4 ℃ overnight; DPBS was washed three times, 5 minutes each, before incubation of the secondary antibody, to wash off excess primary antibody. The secondary antibody mixed solution is DAPI, secondary antibodies which are of different dilution ratios and are resistant to primary antibodies of different excitation wavelengths, 5% serum and DPBS which is supplemented to a required volume, the secondary antibody incubation time is 45 minutes at room temperature, and the DPBS is washed three times for 5 minutes each time. One side of the cells was mounted on a coverslip on a slide. After air-drying the caplets, fluorescence photography can be performed.
3.4, fluorescent shooting
Fluorescence data acquisition was performed using an Olympus inverted fluorescence microscope.
Wherein, the experimental samples are respectively incubated by using primary antibodies FOXA2, 0TX2, HOXB1 and HOXB4, the experimental samples by using primary antibodies FOXA2, 0TX2 and HOXB4 are subsequently incubated by using secondary antibodies Cy3, and the experimental samples by using primary antibodies HOXB4 are incubated by using secondary antibodies FITC.
Experimental results:
the results of measuring FOXA2 and 0TX2 expression on day 11 of differentiation are shown in FIGS. 2A-D, wherein A-C are samples of 3 pieces of FP cells of which the differentiated cells are FOXA2+, and the statistical results of FOXA2 counting are shown in FIG. 2D, and the ratio of FOXA2+ to FP cells is 94.6.+ -. 12.5%.
FIG. 3 shows that A-C is the hindbrain marker HOXB1, D-E is the hindbrain marker HOXB4, and G-I is the forebrain marker OTX2, which is not expressed.
Example 4 functional verification of SHH secretion
Following the procedure shown in FIG. 4A, conditioned medium CM from example 2 and validated for collection and differentiation into FP cells on day 12, neural precursor cells NP were cultured, and mRNA expression levels of the CM-cultured cells VNP ventral gene NKX6.1 and SHH-associated gene GLI2 were detected using real-time quantitative gene amplification fluorescence, reflecting that human basal lamina cells were functional.
The experimental method comprises the following steps: and (3) RNA sample collection: RNA samples in appropriate cell amounts were extracted using RNA extraction kits.
Removal of genomic DNA and reverse transcription synthesis of first strand cDNA: reverse transcription of 500ng of each sample was accomplished using a TAKARA kit, and samples were diluted 92-fold with RNase-free water.
Real-time quantitative PCR: the reaction system was 20. Mu.L containing 9.2. Mu.L of diluted cDNA, 0.8. Mu.L of 10. Mu.M upstream and downstream primer mix, 10. Mu.L of 2×iTaq-SYBR Green super mix, and the real-time quantitative PCR program was set as follows: heat-activated at 95 ℃ for 30s, then 39 cycles: 95℃for 5 seconds, 60℃for 30 seconds, then 65℃for 5 seconds, 95℃for 5 minutes. Three technical replicates were set for each sample.
And (3) data processing: data processing was performed using the 2- ΔΔct method using GAPDH as an internal reference with relative quantification to obtain the relative expression levels of target genes for each sample, and data statistics and mapping were performed using GraphPad Prism 7.0 software.
Experimental results:
as shown in fig. 4B and 4C, there was a significant difference in mRNA expression levels of NKX6.1 and GLI2 in CM-cultured cells, i.e., np+cm group, compared to neural precursor cell NP (< 0.0001).
In the description of the above specification:
the terms "this embodiment," "an embodiment of the invention," "as shown in … …," "further improved embodiments," and the like, mean that a particular feature, structure, material, or characteristic described in the embodiment or example is included in at least one embodiment or example of the invention; in this specification, a schematic representation of the above terms is not necessarily directed to the same embodiment or example, and the particular features, structures, materials, or characteristics described, etc. may be combined or combined in any suitable manner in any one or more embodiments or examples; furthermore, various embodiments or examples, as well as features of various embodiments or examples, described in this specification may be combined or combined by one of ordinary skill in the art without undue experimentation.
Finally, it should be noted that:
the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting thereof;
although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art will appreciate that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some or all of the technical features thereof, without departing from the spirit of the technical solutions of the embodiments of the present invention, and that insubstantial improvements and modifications or substitutions by one skilled in the art from the disclosure herein are within the scope of the invention as claimed.
Claims (9)
1. A method for obtaining hindbrain floor cells in vitro comprising the steps of:
s1, single-layer adherence culture of undifferentiated embryonic stem cells;
s2, adding a TGFbeta/Wnt/node signal channel inhibitor and a BMP signal channel antagonist into an NIM culture medium on the 0 th day of differentiation;
s3, changing a culture medium on the 1 st day of differentiation, and adding a TGFbeta/Wnt/node signal path inhibitor, a BMP signal path antagonist, a Hedgehog signal path Smoothened protein agonist and a homeobox gene agonist into the NIM culture medium;
s4, changing a culture medium on the 5 th day of differentiation, and adding a BMP signal channel antagonist, a Hedgehog signal channel Smoothened protein agonist and a homeobox gene agonist into the NIM culture medium;
s5, changing a culture medium on the 7 th day of differentiation, and adding a BMP signal channel antagonist and a homeobox gene agonist into the NIM culture medium;
s6, continuing to culture until the cells are differentiated into postbrain floor cells;
the homeobox gene agonist includes retinoid acid.
2. A method of obtaining hindbrain floor cells in vitro according to claim 1, wherein the TGFbeta/Wnt/Nodal signaling pathway inhibitor comprises SB431542, at a concentration of SB431542 in the culture medium of 8-12 μΜ.
3. The method of claim 1, wherein the antagonist of BMP signaling pathway comprises DMH1 and wherein the concentration of DMH1 in the culture medium is 1-2 μm.
4. The method of obtaining postbrain floor cells in vitro according to claim 1, wherein the Hedgehog signaling pathway Smoothened protein agonist comprises purporthamine at a concentration of 1.5 to 2.5 μm in culture medium.
5. The method for obtaining postbrain floor cells in vitro according to claim 1, wherein the concentration of retinoid acid in the culture medium is 1.5-2.5 μΜ.
6. The method for obtaining postbrain floor cells in vitro according to claim 1, wherein the culturing time is 10 to 14 days in step S6.
7. The method for obtaining postbrain floor cells in vitro according to claim 6, wherein the postbrain floor cells obtained in step S6 are capable of secreting SHH, and after culturing for 11 days, FOXA2 positive cells are 94.6±12.5%.
8. A complete set of culture medium for inducing embryonic stem cells into hindbrain floor cells in vitro is characterized by comprising a first culture medium, a second culture medium, a third culture medium and a fourth culture medium,
the first culture medium comprises NIM culture medium, TGFbeta/Wnt/Nodal signaling pathway inhibitor and BMP signaling pathway antagonist;
the second culture medium comprises NIM culture medium, TGFbeta/Wnt/node signal channel inhibitor, BMP signal channel antagonist, hedgehog signal channel Smoothened protein agonist, and homeobox gene agonist;
the third culture medium comprises NIM culture medium, BMP signal pathway antagonist, hedgehog signal pathway Smoothened protein agonist, homeobox gene agonist;
the fourth medium comprises NIM medium, BMP signaling pathway antagonists, homeobox gene agonists.
9. The kit of claim 8, wherein the TGFbeta/Wnt/Nodal signaling pathway inhibitor comprises SB431542, the concentration of SB431542 in the medium being 8-12 μΜ; BMP signaling pathway antagonists include DMH1, the concentration of DMH1 in the medium is 1-2 μΜ; the Hedgehog (Hh) signal path Smoothened protein agonist comprises Purmorphamine, the concentration of Purmorphamine in the culture medium is 1.5-2.5 mu m; the Homeobox gene agonist comprises Retinoic acid, and the concentration of the Retinoic acid in the culture medium is 1.5-2.5 mu m.
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